Team:Montpellier/Peptides

Peptides

Introduction

Antimicrobial peptides have an action against different types of bacteria: but what about the vaginal cells and the vaginal flora ? It’s has been shown that most of antimicrobials peptides (AMPs) are hydrophobics and cationics peptides, they bind to the anionic phospholipids on the outer surface of bacterial cell membrane [1]. The affinity for the mammalian cells is on the contrary really low because the outer surface is composed of zwitterionic phospholipids. As a consequence, AMPs will not hurt the vaginal epithelial cells. Moreover, AMPs have an higher affinity for spermatozoa membranes which are composed of anionic phospholipids such as phosphatidylglycerol and phosphatidylserine in the plasma membrane, and caused the lost of motility of spermatozoa and prevent the fecondation. Lastly, all the AMPs studied are not harmful for the Lactobacilli in the vaginal microbiota [2]. All of this reasons make some AMPs promising targets for being used for contraception.

The different AMPs

LL37

LL-37 is an antimicrobial peptide from human macrophages and leukocytes. It is the only member from the human cathelicidin family. This peptide appears to have very important spermicidal effects. “Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use” [2] LL-37 has shown effect in vivo in mice. Females naturally cycling to estrous phase failed to become pregnant when in injected with LL-37 and sperm. LL-37 also showed in vitro effect on human sperm which makes it a very good potential peptide for contraception. The fact that this peptide is naturally produced by human and moreover in the vagina after intercourse adds to the peptide the proof of harmlessness for the vaginal flora. Lastly this peptide is only 37 amino acids long so it is easy to produce and doesn’t need post translational modification.

Subtilosin A

Subtilosin A (SboA) is an antimicrobial peptide of 32 amino acids naturally produced by Bacillus subtilis. This AMP is a very good candidate since it also is involved in various antimicrobial activity against pathogenic organisms such as Gardnerella vaginalis [3] involved in bacterial vaginosis.

SboA is produced and then secreted by B. subtilis thanks to a very complex operon made of 8 genes called the Alb operon (Figure 1).

Production of Subtilosin A

In B. subtilis, the subtilosin A is produced by the expression of an operon of 8 genes Ffigure 1). However, It is possible to get a correctly folded - and therefore active - protein with only SboA and AlbA genes [4]. Indeed, SboA is the Subtilosin A precursor of 32 amino acids, and Alba is part of radical SAM proteins superfamily proteins that use [4Fe-4S]+ cluster to reductively cleave S-adenosyl-L-methionine (SAM) to generate a radical and catalyze the formation of 3 thioether bonds in SboA making it mature.

Iron Sulfur Cluster Complementation

It’s known that the production of mature SboA is more efficient when the SUF genes (Iron Sulfur Cluster) are added to the host organism. It is due to the [4Fe-4S]+ cluster, a cofactor of AlbA [4]. These SUF genes are expressed by numerous prokaryotic cells under stress conditions. And this is no exception of B. subtilis. Also, to make sure that our peptide is well produced and in a sufficient amount, we added the suf genes of B. subtilis to our construction, so that the cluster is produced even in non stressing conditions.

Lacticin 3147

This Class I bacteriocin is composed by two peptides lantibiotic: Ltn⍺ and Ltnbeta. It’s produced by Lactoccocus lactis subsp. lactis [5]. This bacterium contains a 60,232 bp conjugative plasmide, pMRC01 which presents the 6 gene cluster (ltnA1, A2, M1, T and M2) to synthesize lacticin 3147 [6].

ltnA1 and ltnA2 encode LtnA1 and LtnA2 who in their mature active form are termed Ltn⍺ and Ltnꞵ. It was study that ltnM1 and ltnM2 show homology to genes involved in the production and export of lantibiotics. That’s why we used ltnA1, ltnA2, ltnM1 and ltnM2 in our circuit [7]. Some experiments have proved lacticin exhibit spermicidal activity against horse/pony, bovine, boar and rat sperm immobilizing them within 30 s by using peptides LtnA1 and Ltn2 together.

Nisin

Nisin, a 34 amino acid cationic peptide, is a member of the lantibiotic family known to be produced by bacteria, Lactococcus lactis. The encoded precursor is 57 amino acids long, with a 23-residue leader region and a 34-residue structural region.

Thus, the protein Nisin is an effective spermicidal compound with a dose- and time-dependent effect. At a 300–400-g dose, complete immobilization of human spermatozoa was observed within 20 s. Moreover, we know that if we add Nisin in mice during 14 days, we do not see abnormalities in cycle or morphology of the vaginal epithelial cells, inflammatory reaction(s) in the vaginal epithelium [8] . However we don’t know how the Nisin immobilize sperm.

References
[1]	Nguyen LT1, Haney EF, Vogel HJ. (2011). The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol Vol 29, 464–472..
[2]	Tanphaichitr, Nongnuj et al. (2018). Potential Use of Antimicrobial Peptides as Vaginal Spermicides/Microbicides. Pharmaceuticals 9.1 (2016): 13.
[3]	Cavera, V. L., Volski, A., & Chikindas, M. L. (2015). The natural antimicrobial subtilosin A synergizes with lauramide arginine ethyl ester (LAE), ε-poly-l-lysine (polylysine), clindamycin phosphate and metronidazole, against the vaginal pathogen Gardnerella vaginalis. Probiotics and antimicrobial proteins. 7(2), 164-171.
[4]	Paul M. Himes, Scott E. Allen, Sungwon Hwang, and Albert A. Bowers. (2016).Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis. ACS Chemical Biology. 11 (6), 1737-1744.
[5]	Srinivas Suda et al., (2012). Lacticin 3147 - Biosynthesis, Molecular Analysis, Immunity, Bioengineering and Applications. Current Protein & Peptide Science Antimicrob Agents Chemother volume 13, pages 193-204.
[6]	Dougherty et al., (1998). Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147 Mol. Microbiol. 29 (4), 10291038
[7]	Silkin, L. et al., (2008). Spermicidal bacteriocins: Lacticin 3147 and subtilosin A Bioorganic & Medicinal Chemistry Letters 18 3103–3106 Spermicidal
[8]	K V R Reddy, C Aranha, S M Gupta and R D Yedery. (2004). Evaluation of antimicrobial peptide nisin as a safe vaginal contraceptive agent in rabbits: in vitro and in vivo studies. Reproduction Volume 128. Page(s): 117–126.